3.5 PORCENTAJES DEL CHOCOLATE
3.8.4 ESCULPIR Y TALLAR
x_ -
2 r / 8 XO and taking logarithms.In ( x/xo) - (In 2)t/g.
(In x - In xo)/t 0.693/g
H
-1
. dx - In 2 - 0.693x dt g g
H
is called the specific growth rate constant. It is the rate of increase in cell numbers per unit of cell numbers.The steady state environment necessary to study segregational plasmid stability within cells was achieved by the fact that the rate of change in each and every environmental parameter is linked directly to the rate of change of biomass concentration (that is, to the growth rate of the organism). Therefore, all one needs to do is add fresh medium to the culture at a rate sufficient to maintain the culture
population density at some prescribed submaximal value, thereby
replenishing nutrients that are being consumed and, simultaneously,
diluted out end-products that are accumulating at rates exactly
sufficient to ensure that the environment no longer varies with time. The culture was run as an open system, employing an overflow tube (or weir) into the culture so that excess culture can flow from the growth vessel at the same rate that the fresh medium was added, thereby maintaining the culture volume constant.
The construction of a fully defined medium whose composition allows one nutrient to be present at a concentration such that in a batch culture, it would become depleted before any other nutrient was fully used up, has been achieved. Thereby the rate of supply of that nutrient, added along with the other essential nutrients in the fresh medium, would prescribe the rate of growth of the organism in the culture.
Within such nutrient-limited cultures it would be the concentrstion of nutrients in the culture extrscellulsr fluids thst is actually limiting the rate of cell synthesis, since the rate at which such a substrate is taken into the cell will be a function of its concentration.
If the uptake process involves some enzyme-catalysed
reaction, then one might expect that the relationship between uptake rate (and growth rate) and concentration of limiting substrate would be of the form of a Michaelis-Menton equation.
That is
V - Vmax or
M “ /¿max
where V - rate of penetration of substrate.
Vmax - maximum rate of substrate uptake S - concentration of substrate
Kg ■ saturation constant
¡1
■ specific growth rate/imax
■ maximum growth rate Kg - saturation constantMonod (1949) found experimentally that the above relationship did apply
to many culturea, and though it may not be valid under all
circumstances, it forms the cornerstone of much of the theory of
microbial growth in continuous culture, as applied to a chemostat. The change in concentration of the organism with time will depend on the balance between the growth rate
H
and the dilution rate D.Change ■ Growth - Washout
dx - x - Dx
dt
Therefore at equilibrium when dx - o dt M - D
steady state conditions are ultimately established. Where the specific growth rate (/4) and the dilution rate (D) are equal, with the dilution rate set below a critical value at which the organism express their maximum growth rate.
Increasing the dilution rate will increase the specific growth rate of the organism. A point will be reached at which the growth rate will no longer be able to keep pace with the dilution rate at
fimax,
this is termed the maximum specific growth rate and the point at which washout occurs.1.12 Aims of Thesis
The main aim of this thesis was to obtain high expression of the Pseudomonas derived enzyme carboxypeptidae G2 (CPG2) in E. coli and scale up its expression for production.
CPG2 belongs to a class of enzymes which hydrolyse the C- terminal glutamate moiety from folic acid and its analogs (Sherwood et. a l . . 1985). The key role of reduced folates as co-enzymes in many
biochemical pathways has led to the folic acid molecule becoming a prime target in cancer chemotherapy (Bertino et. a l . . 1971). Folate
depletion has most commonly been achieved using the folic acid
antagonist methotrexate to inhibit dihydrofolate reductase (Bleyer, 1978), but may also be achieved directly using the carboxypeptidase G class of enzyme (Kalghatgly and Bertino, 1981). In addition, these
enzymes may be used in rescue therapy following high dosage
methotrexate regimes (Chabner et al., 1972). More recently an antibody - CPG2 conjugate has been shown to be cytotoxic to a choriocarcinoma cell line in vitro (Searle et al., 1986), and to be selectively targeted to tumour tissue in a choriocarcinoma xenograft in nude mice (Melton et a l ., 1986). The potential of CPG2 in cancer chemotherapy has brought forward the requirement for large quantities of purified protein for experimental and clinical testing. T o alleviate this situation the gene encoding CPG2 (cpg) was cloned in E. coli and its entire nucleotide sequence determined (Fig.1.6) (Minton et al.. 1983a; Minton £ t al_., 1984). Expression was obtained in E. coli from a 2.03 kb BamHl fragment of the cpg gene subcloned into the BamHl site of pAT153. This construction in the host W5445 directed expression of CPG2 to between 1Z and 2.3Z of the cells soluble protein into the periplasmic space. The relatively high level of expression, compared to the native promoter (0.2Z) was due to transcriptional read through from the promoter of the upstream tet gene. The expression vector
pNM21 in the host W5445, had exhibited plasmid segregational
instability during cultivation. This was therefore the starting point to improve CPG2 expression. Other factors affecting expression were also investigated; copy number and a temperature inducible promoter system.
fl*» 1.6
Nucleotide Sequence of the cpg gene.
Dashed overlining represents an amino terminal sequence determination in an automated spining cup sequencer. Solid overlining represents elastase peptides which have been completely sequenced and fitted to
the translation DNA sequence. Solid-dotted overllning represents
peptides which have been fitted on the basis of the partial sequencing (solid) and amino acid analysis (dotted). S.D. refers to the putative Shine-Dalgarno sequence (or ribosome-binding site). Possible promoter sequences (i.e. -35 and -10 regions) are indicated by the s o lid lines above and below the relevent sequence. The signal peptide is indicated
by the heavy arrows, as subdivided into its hydrophillic and
i AcççÂççôçiçi
COCA CToc
A A r r CaCT'ATAACa ACCA TOCCT COCCA CCCCCC A CT A CAPOCCI CCA CCACCT TTTTT CA T T CCG A CA A CCCC A AOC A ACA ATCCCT A C.AC.CAOCACA TTCC